title: Identification of amino acid residues involved in the interaction of canine IgE with canine and human FcεRIα.
abstract: The interaction of immunoglobulin E (IgE) antibodies with the high-affinity receptor (FcεRI) is important in anti-parasitic immunity and plays a central role in allergic responses. It has been shown that the human Cε3 domains comprise the binding sites for FcεRIα and crystal structure determination has shown that amino acids in four sites contribute to the high affinity of the interaction. The role of homologous residues within canine IgE-Fc, i.e. amino acids located at Cε2-Cε3 interface (residues 332-337), loop BC (residues 362-365), loop DE (residues 393-396), and loop FG (residues 424-427) in canine Cε3 domain were targeted by site-specific mutagenesis. The functional consequences of the mutations to support (i) IgE-mediated, antigen-induced release of β-hexosaminidase from RBL cells transfected with canine or human FcεRIα and (ii) the affinity of the mutants for the soluble extracellular domain of the α-chain expressed in Pichia pastoris were determined by Surface Plasmon Resonance (SPR). Kinetic analysis supports the observed effects of IgE mutations on stimulus secretion coupling. Potential applications of this study, leading to the generation of an IgE variant with a disabled FcεRIα binding site, are discussed.
authors.affiliation: Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom. Electronic address: mbp06hy@yahoo.co.uk.
authors.forename: Hongtu
authors.initials: H
authors.lastname: Ye
authors.forename: Jonathan E M
authors.initials: JE
authors.lastname: Housden
authors.forename: Michael
authors.initials: M
authors.lastname: Hunter
authors.forename: Sari
authors.initials: S
authors.lastname: Sabban
authors.forename: Birgit A
authors.initials: BA
authors.lastname: Helm
mesh_terms: D000596:Amino Acids / Q000235:genetics; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D004285:Dogs; D014644:Genetic Variation / Q000276:immunology; D006801:Humans; D007073:Immunoglobulin E / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics; D017434:Protein Structure, Tertiary; D051381:Rats; D017455:Receptors, IgE / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D014162:Transfection; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
chemical_list: D000596:Amino Acids; C543355:FcepsilonRI alpha-chain, human; D017455:Receptors, IgE; D007073:Immunoglobulin E; D001619:beta-N-Acetylhexosaminidases
keywords: Canine (ca); Canine IgE variants; FcɛRIα; Human (hu); Human/canine high-affinity receptor alpha chain; IgE/FcɛRIα; Kinetic constants; Soluble (s)